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Query: UNIPROT:P06889 (Mol)
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Previously, we isolated two cDNA clones, TBPOs-1 and TBPOs-2, encoding putative ATPases that are the rice homologues of human immunodeficiency virus-1 (HIV-1) Tat binding protein-1 and subunit 4 of human 26S proteasome. In order to determine the RNA-dependent ATPase activity of these putative proteins, the subclones from these cDNA clones were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant proteins stimulated ATP hydrolysis in the presence of poly(U) and rice total RNA. In contrast, single- and double-stranded forms of HindIII-digested lambda phage DNA are less effective at stimulating ATP hydrolysis. Western blot analysis using antisera against the TBPOs proteins showed a widespread appearance of these proteins in rice tissues and cultured cells. The TBPOs proteins were also found around the region where rice proteasomes would sediment. In addition, the TBPOs-1 protein bound to tobacco TATA-binding protein in vitro. Thus, we suggest that the TBPOs proteins are novel RNA-dependent ATPases characteristic of DEAD-box proteins and propose that the TPBOs proteins can exist in rice proteasomes. Further, the TBPOs-1 protein is thought to play a role in transcriptional events.
Plant Mol Biol 1998 Jun
PMID:Biochemical and immunological characterization of rice homologues of the human immunodeficiency virus-1 Tat binding protein and subunit 4 of human 26S proteasome subunits. 961 16

RNA helicases represent a large family of proteins that have been detected in almost all biological systems where RNA plays a central role. They are ubiquitously distributed over a wide range of organisms and are involved in nuclear and mitochondrial splicing processes, RNA editing, rRNA processing, translation initiation, nuclear mRNA export, and mRNA degradation. RNA helicases are described as essential factors in cell development and differentiation, and some of them play a role in transcription and replication of viral single-stranded RNA genomes. Comparisons of the conserved sequences reveal a close relationship between them and suggest that these proteins might be derived from a common ancestor. Biochemical studies have revealed a strong dependence of the unwinding activity on ATP hydrolysis. Although RNA helicase activity has only been demonstrated for a few examples yet, it is generally believed that all members of the largest subgroups, the DEAD and DEAH box proteins, exhibit this activity.
Crit Rev Biochem Mol Biol 1998
PMID:The protein family of RNA helicases. 974 70

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of approximately 1,400-nt 3'-extended forms (snR13R) and a 108-nt 5'-truncated form (snR13T) that is missing 16 nt at the 5' end. A subpopulation of snR13R contains the same 5' truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5' end and promotes maturation of the 3' end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5' end of snR13T and the 3' end of snR13F reside within C2U4 sequences that immediately flank the C and D boxes. A mutation in the 5' C2U4 repeat causes underaccumulation of snR13F, whereas mutations in the 3' C2U4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3' C2U4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3' C2U4 sequence act in a common pathway to facilitate 3' end formation. Based on these findings, we propose that Sen1p and the C2U4 repeats that flank the C and D boxes promote maturation of the 3' terminus and stability of the 5' terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.
Mol Cell Biol 1998 Dec
PMID:The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae. 981 77

We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria. Using an antiserum raised against extracellular proteins from the pig intestinal isolate L. reuteri 1063, we screened a genomic lambda library derived from the same strain. Affinity purification of recombinant protein from the isolated lambda clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria. Subcloning and introduction of the corresponding gene, here denoted aggHinto the L. reuteri type strain markedly enhanced aggregation. Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation-deficient phenotype. Finally, an affinity-purified and cleaved fusion of AggH protein and the maltose-binding protein, MBP, strongly promoted aggregation of L. reuteri 1063, whereas the uncleaved fusion protein was inactive. Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP-dependent DEAD-box helicases. These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation.
Mol Microbiol 1999 Apr
PMID:Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD-box helicase. 1023 97

To examine whether two DEAD box genes, DDX1 and DDX6, would have some roles in the progression of tumors, we investigated the correlation of the expression of these genes with that of MYCN in neuroblastomas either with or without MYCN amplification. The mRNA of MYCN was observed only in the cell lines with amplification of MYCN. The mRNAs of DDX1 and DDX6 were found in all the cell lines examined, but the correlation between the mRNA levels of DDX1 or DDX6 and MYCN was poor. These findings suggest that the expression of neither DEAD box gene is correlated with the gene expression of MYCN.
Biochem Mol Biol Int 1999 Apr
PMID:Expression of two dead box genes (DDX1 and DDX6) is independent of that of MYCN in human neuroblastoma cell lines. 1031 7

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.
Mol Microbiol 1999 May
PMID:Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation. 1036 Dec 80

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.
Mol Cell Biol 1999 Sep
PMID:RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. 1045 74

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.
J Mol Biol 1999 Oct 01
PMID:Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA. 1052 3

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.
J Mol Biol 1999 Oct 01
PMID:The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI. 1052 5

DEAD-box proteins have been implicated in a wide array of cellular processes ranging from initiation of protein synthesis and ribosome biogenesis to mRNA splicing. Here, we report the isolation, biochemical characterization and crystallization of the first thermophilic DEAD box protein, Hera (heat-resistant RNA-dependent ATPase) from Thermus thermophilus HB8. The molecular mass of the deduced Hera protein sequence (510 amino acid residues) is 55.95 kDa. Hera possesses all of the conserved motifs found among the, DEAD-box RNA helicases. In addition, it also has a motif characteristic of the protein component of ribonuclease P at its C-terminal region (residues 372-386). Hera appears to be non-specific with respect to the RNA species that triggers ATPase activity. Nevertheless, at high temperature, ATPase activity is at a maximum when bacterial 16 S rRNA or 23 S rRNA are used as the substrates. Moreover, a deletion of the RNase P protein motif significantly reduces the ability of Hera to hydrolyze ATP in the presence of RNase P RNA. Hera has a specific ATPase activity of 480 units/microg and therefore, displays the highest ATPase specific activity reported for a protein of the RNA helicase family. We determined that Hera shows helix-destabilizing activity, and that the RNA-unwinding or helix-destabilizing activity of Hera is coupled to ATP hydrolysis. Since Hera is a stable thermophilic protein and we have obtained crystals of it diffracting beyond 2.6 A, the possibilities for structure determination of a full-length RNA-helicase are open.
J Mol Biol 1999 Dec 03
PMID:Hera from Thermus thermophilus: the first thermostable DEAD-box helicase with an RNase P protein motif. 1061 Jul 97


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